Proteomic characterization of oxidative dysfunction in human umbilical vein endothelial cells (HUVEC) induced by exposure to oxidized LDL

The oxidative modification of low-density lipoprotein (LDL) and subsequent alteration of endothelial cell function are generally accepted as an important early event in the pathogenesis of atherosclerosis. To understand the mechanism by which oxidized LDL (oxLDL) causes dysfunction in endothelial cells, human umbilical vein endothelial cells (HUVEC) were exposed to oxLDL at a concentration that induces cellular dysfunction, and proteomic analysis was carried out, together with the analysis of cellular lipid peroxidation products. Time-dependent accumulation of 7-ketocholesterol and the progression of oxidative modification of peroxiredoxin 2 were observed, together with the suppression of cell proliferation. Proteomic analysis using two-dimensional gel electrophoresis (2-D gel) revealed that nucleophosmin, stathmin, and nucleolin were differentially expressed after exposure to oxLDL. Both 2-D gel and western blot analyses revealed that (1) nucleophosmin was dephosphorylated in a time-dependent manner; (2) stathmin was transiently phosphorylated at 6 h, and the unphosphorylated form was continuously down-regulated; and (3) nucleolin was identified as a 20-kDa fragment and a 76-kDa form, which were down-regulated. These observations suggest that the exposure of HUVEC to oxLDL results in the suppression of cell proliferation, which is ascribed to protein modification and/or altered expression of nucleophosmin, stathmin, and nucleolin under these oxidative stress conditions.     

Cigarette smoke and LDL cooperate in reducing nitric oxide bioavailability in endothelial cells via effects on both eNOS and NADPH oxidase

The ubiquitous free radical nitric oxide (NO) plays an important role in many biological processes, including the regulation of both vascular tone and inflammatory response; however, its role in the effects of cigarette smoke exposure on atherosclerosis remains unclear. Our aim was to study the mechanisms of NO regulation in endothelial cells in response to cigarette smoke exposure in vitro. Using human umbilical vein endothelial cells (HUVEC), we have demonstrated that combining non-toxic concentrations of cigarette smoke bubbled through PBS (smoke-bubbled PBS [sbPBS]) with native LDL (nLDL) significantly reduces the amount of bioavailable NO. The effect is comparable to that seen with oxidized LDL (oxLDL), but has not been seen with sbPBS or nLDL alone. Mechanistic investigations showed that the combination of sbPBS+nLDL did not reduce the amount of endothelial nitric oxide synthase (eNOS), but did inhibit its enzymatic activity. Concomitantly, both sbPBS+nLDL and oxLDL significantly increased the production of reactive oxygen species (ROS) in the form of superoxide anions (()O(2)(-)) and peroxynitrite (ONOO(-)) in HUVEC. Selective inhibition of NADPH oxidase prevented this response. Incubation of sbPBS+nLDL revealed the formation of 7-ketocholesterol (7-KC) and 7-hydroxycholesterol, which are indicators for oxidative modification of LDL. This could explain the reported increase in circulatory levels of oxLDL in smokers. Our results suggest that reduction of functional NO in response to a combination of sbPBS+nLDL is secondary to both reduction of eNOS activity and stimulation of NADPH oxidase activity. Because sbPBS alone showed no effect on eNOS activity or ROS formation, nLDL should be included in cigarette-smoke-related mechanistic in vitro experiments on endothelial cells to be more reflective of the clinical situation.     

Oxidized LDL up-regulates the ascorbic acid transporter SVCT2 in endothelial cells

Endothelial dysfunction is an early manifestation of atherosclerosis caused in part by oxidized LDL (oxLDL). Since vitamin C, or ascorbic acid, prevents several aspects of endothelial dysfunction, the effects of oxLDL on oxidative stress and regulation of the ascorbate transporter, SVCT2, were studied in cultured EA.hy926 endothelial cells. Cells cultured for 18 h with 0.2 mg/ml oxLDL showed increased lipid peroxidation that was prevented by a single addition of 0.25 mM ascorbate at the beginning of the incubation. This protection caused a decrease in intracellular ascorbate, but no change in the cell content of GSH. In the absence of ascorbate, oxLDL increased SVCT2 protein and function during 18 h in culture. Although culture of the cells with ascorbate did not affect SVCT2 protein expression, the oxLDL-induced increase in SVCT2 protein expression was prevented by ascorbate. These results suggest that up-regulation of endothelial cell SVCT2 expression and function may help to maintain intracellular ascorbate during oxLDL-induced oxidative stress, and that ascorbate in turn can prevent this effect.     

Protein modification elicited by oxidized low-density lipoprotein (LDL) in endothelial cells: protection by (-)-epicatechin

The action of oxidatively modified low-density lipoprotein (oxLDL) on vascular endothelial cells has been proposed to be a crucial process leading to endothelial dysfunction and atherogenesis. OxLDL was shown here to elicit oxidative stress in bovine aortic endothelial cells or human umbilical vein endothelial cells, as judged by an increase in 2',7'-dichlorofluorescein fluorescence and elevated levels of carbonylated, nitrated, and 2-hydroxynonenal-coupled proteins. These effects were sensitive to apocynin, indicating involvement of NADPH oxidase. A 170-kDa polypeptide carbonylated upon exposure of cells to oxLDL was identified by immunoprecipitation as EGF receptor. Immunocytochemical visualization by confocal microscopy revealed the highest levels of modified proteins in the perinuclear region. Exposure of endothelial cells to oxLDL led to modulation of the expression levels of *NO synthases; the endothelial isoform (eNOS) was down-regulated via proteasomal degradation, whereas the inducible isoform (iNOS) was up-regulated in an enzymatically active state. eNOS protein was found to be both carbonylated and nitrated upon exposure of cells to oxLDL. iNOS contributed to the generation of modified proteins as judged by the effects of the selective inhibitor L-NIO. These oxLDL-elicited changes in vascular endothelial cells described were suppressed by (-)-epicatechin, a dietary polyphenol, which inhibited NADPH oxidase activity in these cells.     

Protective effects of honokiol against oxidized LDL-induced cytotoxicity and adhesion molecule expression in endothelial cells

Honokiol, a compound extracted from Chinese medicinal herb Magnolia officinalis, has several biological effects. However, its protective effects against endothelial injury remain unclarified. In this study, we examined whether honokiol prevented oxidized low-density lipoprotein (oxLDL)-induced vascular endothelial dysfunction. Incubation of oxLDL with honokiol (2.5-20 microM) inhibited copper-induced oxidative modification as demonstrated by diene formation, thiobarbituric acid reactive substances (TBARS) assay and electrophoretic mobility assay. Expression of adhesion molecules (ICAM, VCAM and E-selectin) and endothelial NO synthase (eNOS) affected by oxLDL was investigated by flow cytometry and Western blot. We also measured the production of reactive oxygen species (ROS) using the fluorescent probe 2',7'-dichlorofluorescein acetoxymethyl ester (DCF-AM). Furthermore, several apoptotic phenomena including increased cytosolic calcium, alteration of mitochondrial membrane potential, cytochrome c release and activation of caspase-3 were also investigated. Apoptotic cell death was characterized by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stain. The results showed that honokiol prevented the copper-induced oxidative modification of LDL. Honokiol also ameliorated the oxLDL-diminished eNOS protein expression and reduced the oxLDL-induced adhesion molecules and the adherence of THP-1 cells to HUVECs. Furthermore, honokiol attenuated the oxLDL-induced cytotoxicity, apoptotic features, ROS generation, intracellular calcium accumulation and the subsequent mitochondrial membrane potential collapse, cytochrome c release and activation of caspase-3. Our results suggest that honokiol may have clinical implications in the prevention of atherosclerotic vascular disease.     

Modified LDLs induce proliferation-mediated death of human vascular endothelial cells through MAPK pathway

The ability of modified low-density lipoptoteins (LDLs) to induce both proliferation and death of endothelial cells is considered to be a mechanism of early atherosclerosis development. We previously showed that carbamylated LDL (cLDL) induces human coronary artery endothelial cell (HCAEC) death in vitro. This effect is similar to the atherogenic action of oxidized LDL (oxLDL) that induces the proliferation and death of endothelial cells. The present study was designed to analyze a potential proliferative effect of cLDL and whether proliferation caused by modified LDLs is related to cell death. Cultured HCAECs were exposed to different concentrations of modified LDL or native LDL for varying periods of time. Cell proliferation measured by bromodeoxyuridine incorporation and S-phase analysis was dose-dependently increased in the presence of cLDL (6.25-200 microg/ml). The proliferation induced by cLDL or oxLDL was associated with cell death and increased phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK). Inhibition of cLDL- or oxLDL-induced proliferation by aphidicolin (1 microg/ml) was protective against both short-term cell death measured by lactate dehydrogenase release into the medium and long-term cell viability visualized by cell multiplication. Inhibition of ERK phosphorylation led to a significant decrease of DNA synthesis and cell rescue from injury by modified LDLs, while inhibition of JNK phosphorylation had an only partial rescue effect without involvement in cell proliferation. These data are the first evidence that endothelial cell death induced by cLDL or oxLDL is mediated by cell proliferation through the mitogen-activated protein kinase pathway.     

Adiponectin suppresses proliferation and superoxide generation and enhances eNOS activity in endothelial cells treated with oxidized LDL

Adiponectin (also known as 30-kDa adipocyte complement-related protein or Acrp30) is an abundant adipocyte-derived plasma protein with anti-atherosclerotic and insulin-sensitizing properties. In order to investigate the potential mechanism(s) of the vascular protective effect of adiponectin, we used cultured bovine endothelial cells (BAECs) to study the effect of recombinant globular adiponectin (gAd) on cellular proliferation and the generation of reactive oxygen species (ROS) induced by oxidized LDL (oxLDL). By RT-PCR, we found that BAECs preferentially express AdipoR1, the high-affinity receptor for gAd. Treatment of BAECs with oxLDL (10 μg/ml) for 16 h stimulated cell proliferation by ∼60%, which was inhibited by co-incubation with gAd. Cell treatment with gAd also inhibited basal and oxLDL-induced superoxide release, and suppressed the activation of p42/p44 MAP kinase by oxLDL. The effects of gAd were blocked by a specific polyclonal anti-adiponectin antibody (TJ414). OxLDL-induced BAEC proliferation and superoxide release were inhibited by the NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI), but not the eNOS inhibitor l-nitroarginine methyl ester (l-NAME). Finally, gAd ameliorated the suppression of eNOS activity by oxLDL. These data indicate that gAd inhibits oxLDL-induced cell proliferation and suppresses cellular superoxide generation, possibly through an NAD(P)H oxidase-linked mechanism.     

The Variability of oxLDL-induced Cytotoxicity on Different Types of Cell Lines

The epidemiologic studies indicated an association of obesity with increased incidence of colorectal, breast and ovarian cancer. Further studies found a positive correlation between increased serum oxLDL and an increased risk of the three cancers. In contrast, our previous studies found a negative correlation between the serum oxLDL levels and the risk of leukemia and esophageal cancer. Identification of the variability of cytotoxicity of oxLDL-induced on different types of cell lines is important for understanding the mechanism of oxLDL involved in the tumorigenesis. In the present study, we investigated the effective impacts of oxLDL on the proliferation and apoptosis for the human umbilical vein endothelial cells (HUVEC) and two cancer cell lines (EC-9706 and K562/AO2 with multi-drug resistance). HUVEC, K562/AO2 and EC-9706 cell lines were cultured in the presence of oxLDL, and cell proliferation was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, apoptosis and cell cycle by flow cytometer, mRNA expression by RT-PCR and protein expression by Western blot. OxLDL could inhibit proliferation and apoptosis of the three cell lines; however, there were significant differences of effective action on the viability and apoptosis. The dose of oxLDL-induced cytotoxicity on HUVEC was higher than that on the two tumor cells. The antibody of lectin-like oxLDL receptor-1 (LOX-1-ab) can block oxLDL-induced cytotoxicity. Cells apoptosis is mediated by reducing Bcl-2 and increasing Bax and caspase-3 mRNA and protein expression. This study showed the dose of oxLDL-induced cytotoxicity on HUVEC was higher than that on K562/AO2 and EC-9706 tumor cells. The antibody of LOX-1 receptor can block the oxLDL-induced cytotoxicity.     

Oxidized low-density lipoprotein and 15-deoxy-delta 12,14-PGJ2 increase mitochondrial complex I activity in endothelial cells

Oxidized lipids are capable of initiating diverse cellular responses through both receptor-mediated mechanisms and direct posttranslational modification of proteins. Typically, exposure of cells to low concentrations of oxidized lipids induces cytoprotective pathways, whereas high concentrations result in apoptosis. Interestingly, mitochondria can contribute to processes that result in either cytoprotection or cell death. The role of antioxidant defenses such as glutathione in adaptation to stress has been established, but the potential interaction with mitochondrial function is unknown and is examined in this article. Human umbilical vein endothelial cells (HUVEC) were exposed to oxidized LDL (oxLDL) or the electrophilic cyclopentenone 15-deoxy-Delta 12,14-PGJ2 (15d-PGJ2). We demonstrate that complex I activity, but not citrate synthase or cytochrome-c oxidase, is significantly induced by oxLDL and 15d-PGJ2. The mechanism is not clear at present but is independent of the induction of GSH, peroxisome proliferator-activated receptor (PPAR)-gamma, and PPAR-alpha. This response is dependent on the induction of oxidative stress in the cells because it can be prevented by nitric oxide, probucol, and the SOD mimetic manganese(III) tetrakis(4-benzoic acid) porphyrin chloride. This increased complex I activity appears to contribute to protection against apoptosis induced by 4-hydroxynonenal.     

The essential role of p38 MAPK in mediating the interplay of oxLDL and IL-10 in regulating endothelial cell apoptosis

Interleukin-10 (IL-10) may have therapeutic potential in various inflammatory diseases, including atherosclerosis, as it can inhibit oxLDL-induced foam cell formation and apoptosis in macrophages. This study investigated the effect of IL-10 on mitogen-activated protein kinase (MAPK) activation, and apoptosis induced by oxidized low-density lipoprotein (oxLDL) in cultured human umbilical vein endothelial cells (HUVEC). The results demonstrated that IL-10 significantly blocked the phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) and apoptosis induced by oxLDL. The inhibitory effect of IL-10 on oxLDL-induced apoptosis was partially dependent on reduced p38, but not JNK, phosphorylation. This study also discovered a linkage between IL-10 and p38 MAPK signaling in oxLDL-induced endothelial cell apoptosis. Interestingly, this study found that lectin-like oxidized LDL receptor-1 (LOX-1) was the only scavenger receptor, on the surface of HUVEC, that was upregulated by oxLDL and the increase in LOX-1 was not suppressed by IL-10. This study confirmed that IL-10 significantly upregulated the expression of suppressor of cytokine signaling-3 (SOCS3), whereas SOCS3 knockdown by siRNA effectively blocked the inhibitory effect of IL-10 on p38 MAPK-dependent apoptosis induced by oxLDL. These results showed for the first time, that IL-10 modulated oxLDL-induced apoptosis by upregulating SOCS3, which then interrupted p38 MAPK activation in endothelial cells. These findings support the essential role of p38 MAPK in the interplay of oxLDL and IL-10 in endothelial apoptosis.     

Proteomic approach to study the effects of various oxidatively modified low-density lipoprotein on regulation of protein expression in human umbilical vein endothelial cell

Circulating low-density lipoprotein (LDL) isolated by our laboratory, a new form of modified LDL and designated as L5, has been reported to be cytotoxic by inducing apoptosis of vascular endothelial cells in vitro. The objective of this study was to compare the biological functions of three different forms of oxidatively modified LDL on human umbilical vein endothelial cells (HUVEC) by proteomic approaches. HUVEC were incubated with serum-free medium, native LDL (N-LDL), L5 isolated from familial hypercholesterolemic subjects (FH-L5), copper-oxidized LDL (Cu-ox-LDL), and atheroma-derived LDL (a-LDL) at 37 °C for 24 h. We found that HUVEC incubated with FH-L5 expressed approximately 3 fold higher concentration of MCP-1 than did cells subject to other treatments. All modified LDL significantly suppressed ATP synthase, Grp58, Grp78, and Prdx3. However, the expression of hnRNP C1/C2 was significantly enhanced by FH-L5 and a-LDL; glutathione transferase was significantly enhanced only by FH-L5. A concordant pattern of protein expression was observed between immunoblotting and 2D electrophoresis. Different forms of oxidatively modified LDL regulated HUVEC protein expression in different patterns, suggesting different roles for different oxLDL forms in inducing atherogenesis.     

Induction of the oxLDL receptor LOX-1 by endothelin-1 in human endothelial cells.

In this study, we analyzed the effect of endothelin-1 (ET-1) on expression of the lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 LOX-1 and on oxLDL uptake in primary cultures of human umbilical vein endothelial cells (HUVEC). LOX-1 mRNA was quantified by standard-calibrated competitive RT-PCR, LOX-1 protein expression by Western analysis and endothelial oxLDL uptake using DiI-labeled oxLDL. ET-1 induces LOX-1 mRNA expression, reaching its maximum after 1 h (160 +/- 14% of control, 100 nM ET-1, P < 0.05). This increased ET-1-mediated LOX-1 mRNA expression could be inhibited by endothelin receptor B antagonist BQ-788. In addition, ET-1 stimulates LOX-1 protein expression and oxLDL uptake in HUVEC. The augmented oxLDL uptake by ET-1 is mediated by endothelin receptor B, but not by protein kinases. These data support a new pathophysiological mechanism how locally and systemically increased ET-1 levels could promote LOX-1-mediated oxLDL uptake in human endothelial cells and the development and progression of endothelial dysfunction and atherosclerosis.     

Enzymatic modification of low density lipoprotein by purified lipoxygenase plus phospholipase A2 mimics cell-mediated oxidative modification

Low density lipoprotein (LDL) can be oxidatively modified by cultured endothelial cells or by cupric ions, resulting in increased macrophage uptake of the lipoprotein. This process could be relevant to the formation of macrophage-derived foam cells in the early atherosclerotic lesion. The mechanism of endothelial cell modification of LDL is unknown. In the present work we show that incubation of LDL with purified soybean lipoxygenase, in the presence of pure phospholipase A2, can mimic endothelial cell-induced oxidative modification. Typically, incubation with lipoxygenase plus phospholipase A2 caused: 1) generation of about 15 nmol of thiobarbituric acid-reactive substances per mg of LDL protein; 2) a 4- to 7-fold increase in the rate of subsequent macrophage degradation of the LDL; 3) a 10-fold decrease in recognition by fibroblasts; 4) a marked increase in electrophoretic mobility in agarose gels; and, 5) disappearance of intact apoprotein B on SDS polyacrylamide gels. Degradation of the enzymatically modified LDL by macrophages was competitively inhibited by endothelial cell-modified LDL and by polyinosinic acid, but only partially suppressed by acetylated LDL. The lipoxygenase plus phospholipase A2-induced modification of LDL is not necessarily identical to endothelial cell modification, but it is a useful model for studying the mechanism of oxidative modification of LDL. This work also represents the first example of oxidative modification of LDL by specific enzymes leading to enhanced recognition by macrophages.     

Cytotoxicity of myeloperoxidase/nitrite-oxidized low-density lipoprotein toward endothelial cells is due to a high 7beta-hydroxycholesterol to 7-ketocholesterol ratio

Oxygenated cholesterols (oxysterols) formed during oxidation of low-density lipoprotein (LDL) are associated with endothelial dysfunction and atherogenesis. We compared the profile of oxysterols in modified human LDL obtained on reaction with myeloperoxidase/H2O2 plus nitrite (MPO/H2O2/nitrite-oxLDL) with that on Cu2+ -catalyzed oxidation. The 7beta-hydroxycholesterol/7-ketocholesterol ratio was markedly higher in MPO/H2O2/nitrite-oxLDL than in Cu2+ -oxidized LDL (7.9 +/- 3.0 versus 0.94 +/- 0.10). Like MPO/H2O2/nitrite-oxLDL, 7beta-hydroxycholesterol was cytotoxic toward endothelial cells through eliciting oxidative stress. Cytotoxicity was accompanied by DNA fragmentation and was prevented by the NADPH oxidase inhibitor apocynin, suggesting stimulation of NADPH oxidase-mediated O2-* formation. 7-Ketocholesterol was only cytotoxic when added alone, whereas a 1:1-mixture with 7beta-hydroxycholesterol surprisingly was noncytotoxic. We conclude from our data that (i) 7beta-hydroxycholesterol is a pivotal cytotoxic component of oxidized LDL, (ii) 7-ketocholesterol protects against 7beta-hydroxycholesterol in oxysterol mixtures or oxLDL, (iii) the 7beta-hydroxycholesterol/7-ketocholesterol ratio is a crucial determinant for cytotoxicity of oxidized LDL species and oxysterol mixtures, and (iv) the low share of 7-ketocholesterol explains the higher cytotoxicity of MPO/H2O2/nitrite-oxLDL than other forms of oxidized LDL. The dietary polyphenol (-)-epicatechin inhibited not only formation but also cytotoxic actions of both oxLDL and oxysterols.     

Supression of hemin-mediated oxidation of low-density lipoprotein and subsequent endothelial reactions by hydrogen sulfide (H2S)

Heme-mediated oxidative modification of low-density lipoprotein (LDL) plays a crucial role in early atherogenesis. It has been shown that hydrogen sulfide (H₂S) produced by vascular smooth muscle cells is present in plasma at a concentration of about 50 µmol/L. H₂S is a strong reductant which can react with reactive oxygen species like superoxide anion and hydrogen peroxide. The current study investigated the effect of H₂S on hemin-mediated oxidation of LDL and oxidized LDL (oxLDL)-induced endothelial reactions. H₂S dose dependently delayed the accumulation of lipid peroxidation products—conjugated dienes, lipid hydroperoxides (LOOH), and thiobarbituric acid reactive substances—during hemin-mediated oxidation. Moreover, H₂S decreased the LOOH content of both oxidized LDL and lipid extracts derived from soft atherosclerotic plaque, which was accompanied by reduced cytotoxicity. OxLDL-mediated induction of the oxidative stress responsive gene, heme oxygenase-1, was also abolished by H₂S. Finally we have shown that H₂S can directly protect endothelium against hydrogen peroxide and oxLDL-mediated endothelial cytotoxicity. These results demonstrate novel functions of H₂S in preventing hemin-mediated oxidative modification of LDL, and consequent deleterious effects, suggesting a possible antiatherogenic action of H₂S.     

OxLDL induces endothelial dysfunction and death via TRAF3IP2: Inhibition by HDL3 and AMPK activators

Oxidized low-density lipoprotein (oxLDL) induces endothelial cell death through the activation of NF-κB and AP-1 pathways. TRAF3IP2 is a redox-sensitive cytoplasmic adapter protein and an upstream regulator of IKK/NF-κB and JNK/AP-1. Here we show that oxLDL-induced death in human primary coronary artery endothelial cells (ECs) was markedly attenuated by the knockdown of TRAF3IP2 or the lectin-like oxLDL receptor 1 (LOX-1). Further, oxLDL induced Nox2/superoxide-dependent TRAF3IP2 expression, IKK/p65 and JNK/c-Jun activation, and LOX-1 upregulation, suggesting a reinforcing mechanism. Similarly, the lysolipids present in oxLDL (16:0-LPC and 18:0-LPC) and minimally modified LDL also upregulated TRAF3IP2 expression. Notably, whereas native HDL3 reversed oxLDL-induced TRAF3IP2 expression and cell death, 15-lipoxygenase-modified HDL3 potentiated its proapoptotic effects. The activators of the AMPK/Akt pathway, adiponectin, AICAR, and metformin, attenuated superoxide generation, TRAF3IP2 expression, and oxLDL/TRAF3IP2-mediated EC death. Further, both HDL3 and adiponectin reversed oxLDL/TRAF3IP2-dependent monocyte adhesion to endothelial cells in vitro. Importantly, TRAF3IP2 gene deletion and the AMPK activators reversed oxLDL-induced impaired vasorelaxation ex vivo. These results indicate that oxLDL-induced endothelial cell death and dysfunction are mediated via TRAF3IP2 and that native HDL3 and the AMPK activators inhibit this response. Targeting TRAF3IP2 could potentially inhibit progression of atherosclerotic vascular diseases.